Karin Önnheim

Galectin 3 aggravates joint inflammation and destruction in antigen-induced arthritis

OBJECTIVE Galectin 3, an endogenous β-galactoside-binding lectin, plays an important role in the modulation of immune responses. The finding that galectin 3 is present in the inflamed synovium in patients with rheumatoid arthritis suggests that the protein is associated with the pathogenesis of this disease. We undertook this study to investigate the influence of galectin 3 deficiency in a murine model of arthritis. METHODS Wild-type (WT) and galectin 3-deficient (galectin 3(-/-) ) mice were subjected to antigen-induced arthritis (AIA) through immunization with methylated bovine serum albumin. The concentration of serum cytokines (interleukin-6 [IL-6] and tumor necrosis factor α [TNFα]) and antigen-specific antibodies was evaluated using a cytometric bead array platform and enzyme-linked immunosorbent assay (ELISA). Cellular IL-17 responses were examined by flow cytometry, ELISA, and enzyme-linked immunospot assay. RESULTS The joint inflammation and bone erosion of AIA were markedly suppressed in galectin 3(-/-) mice as compared with WT mice. The reduced arthritis in galectin 3(-/-) mice was accompanied by decreased levels of antigen-specific IgG and proinflammatory cytokines. The frequency of IL-17-producing cells in the spleen was reduced in galectin 3(-/-) mice as compared with WT mice. Exogenously added recombinant galectin 3 could partially restore the reduced arthritis and cytokines in galectin 3(-/-) mice. CONCLUSION Our findings show that galectin 3 plays a pathogenic role in the development and progression of AIA and that the disease severity is accompanied by alterations of antigen-specific IgG levels, systemic levels of TNFα and IL-6, and frequency of IL-17-producing T cells. To our knowledge, this is the first report of in vivo evidence that galectin 3 plays a crucial role in the development of arthritis.

The Host Defense Peptide LL-37 Selectively Permeabilizes Apoptotic Leukocytes

ABSTRACT LL-37 is a cationic host defense peptide that is highly expressed during acute inflammation and that kills bacteria by poorly defined mechanisms, resulting in permeabilization of microbial membranes. High concentrations of LL-37 have also been reported to have cytotoxic effects against eukaryotic cells, but the peptide is clearly capable of differentiating between membranes with different compositions (eukaryotic versus bacterial membranes). Eukaryotic cells such as leukocytes change their membrane composition during apoptotic cell death, when they are turned into nonfunctional but structurally intact entities. We tested whether LL-37 exerted specific activity on apoptotic cells and found that the peptide selectively permeabilized the membranes of apoptotic human leukocytes, leaving viable cells unaffected. This activity was seemingly analogous to the direct microbicidal effect of LL-37, in that it was rapid, independent of known surface receptors and/or active cell signaling, and inhibitable by serum components such as high-density lipoprotein. A similar selective permeabilization of apoptotic cells was recorded for both NK cells and neutrophils. In the latter cell type, LL-37 permeabilized both the plasma and granule membranes, resulting in the release of both lactate dehydrogenase and myeloperoxidase. Apoptosis is a way for inflammatory cells to die silently and minimize collateral tissue damage by retaining tissue-damaging and proinflammatory substances within intact membranes. Permeabilization of apoptotic leukocytes by LL-37, accompanied by the leakage of cytoplasmic as well as intragranular molecules, may thus shift the balance between pro- and anti-inflammatory signals and in this way be of importance for the termination of acute inflammation.

Tumour necrosis factor (TNF)-α primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization

Neutrophil granulocytes play an important role in innate host defence against microbial invasions and they are also the key effector cells in mediating host tissue damage. These functions often rely on the production of reactive oxygen species (ROS) from the membrane‐bound NADPH‐oxidase system. The magnitude of ROS production varies depending on the state of the cells, i.e. resting or primed. Many priming agents as well as potent NADPH‐oxidase activators have been identified and characterized for human neutrophils. The cytokine tumour necrosis factor (TNF)‐α is one prominent example of a priming agent and the synthetic hexapeptide WKYMVm is an agonist that triggers an activation of the NADPH‐oxidase of human neutrophils through two members of the formyl peptide family of receptors, formyl peptide receptor (FPR) and FPR‐like 1 (FPRL1). This peptide also activates murine neutrophils but the precise receptor involved has not been previously characterized. We show in this study that WKYMVm activates stably transfected HL60 cells expressing murine formyl peptide receptor‐related sequence 2 (Fpr‐rs2) and that activation of murine neutrophils with WKYMVm is blocked by an FPRL1‐specific antagonist. WKYMVm is thus an agonist for Fpr‐rs2 and we suggest that this receptor is in fact the mouse orthologue of FPRL1. In addition, we show that the WKYMVm response in murine neutrophils can be primed by TNF‐α and this priming process involves mobilization of subcellular granules. The results obtained using neutrophils derived from TNF receptor type I (TNFRI)‐deficient animals suggest that TNF‐α exerts its priming effect via the TNFRI.

Reactivation of Desensitized Formyl Peptide Receptors by Platelet Activating Factor: A Novel Receptor Cross Talk Mechanism Regulating Neutrophil Superoxide Anion Production

Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPRdes) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPRdes back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPRdes as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPRdes also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPRdes initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.

A novel receptor cross-talk between the ATP receptor P2Y2 and formyl peptide receptors reactivates desensitized neutrophils to produce superoxide.

Neutrophils express several G-protein coupled receptors (GPCRs) and they cross regulate each other. We described a novel cross-talk mechanism in neutrophils, by which signals generated by the receptor for ATP (P2Y2) reactivate desensitized formyl peptide receptors (FPRs) so that these ligand-bound inactive FPRs resume signaling. At the signaling level, the cross-talk was unidirectional, i.e., P2Y2 ligation reactivated FPR, but not vice versa and was sensitive to the phosphatase inhibitor calyculinA. Further, we show that the cross talk between P2Y2 and FPR bypassed cytosolic Ca(2+) transients and did not rely on the actin cytoskeleton. In summary, our data demonstrate a novel cross-talk mechanism that results in reactivation of desensitized FPRs and, an amplification of the neutrophil response to ATP.

The β-galactoside binding immunomodulatory lectin Galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: Role of reactive oxygen species generated by the NADPH-oxidase and inactivation of the agonist

Neutrophils interacting with a chemoattractant gradually become nonresponsive to further stimulation by the same agonist, a process known as desensitization. Receptor desensitization is a highly regulated process that involves different mechanisms depending on which receptor-ligand pair that is studied. Galectin-3, a member of a large family of beta-galactoside-binding lectins, has been suggested to be a regulator of the inflammatory process, augmenting or directly triggering the neutrophil functional repertoire. We show here that the desensitized state of neutrophils interacting with the chemotactic peptide fMLF is broken by galectin-3 and that this is achieved through an oxygen radical-mediated inactivation of the chemoattractant. The effect was inhibited by the competitor lactose and required the affinity of galectin-3 for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins. The latter was shown using a galectin-3 mutant that lacked N-acetyllactosamine binding activity, and this protein was not active. The mechanism behind the inactivation of the chemoattractant was found to depend on the ability of galectin-3 to induce a neutrophil generation/secretion of reactive oxygen species which in combined action with myeloperoxidase inactivated the peptides.

The Subcellular Localization of the Receptor for Platelet-Activating Factor in Neutrophils Affects Signaling and Activation Characteristics

The localization in neutrophils, of the receptor for platelet-activating factor (PAFR), has been determined using subcellular fractionation and a receptor mobilization protocol. We show that the PAFR is expressed primarily in the plasma membrane. Although activation of neutrophils by PAF induces responses typical also of agonists that bind the formyl peptide receptors (FPR), known to be stored in mobilizable organelles, some quantitative as well as qualitative differences were observed when neutrophils were activated through these receptors. PAF is equipotent to fMLF (high affinity agonist for FPR1) to cleave off L-selectin and to induce granule/vesicle secretion but is more potent than fMLF to induce a rise in intracellular Ca2+. Similar to fMLF, PAF induced also a robust release of reactive oxygen species, but with higher EC50 value and was less sensitive to a PI3K inhibitor compared to the fMLF response. Despite the lack of a granule localized storage pool of receptors, the PAF-induced superoxide production could be primed; receptor mobilization was, thus, not required for priming of the PAF response. The desensitized PAFR could not be reactivated, suggesting that distinct signaling pathways are utilized for termination of the responses triggered through FPR1 and PAFR.

Vaccination with the Secreted Glycoprotein G of Herpes Simplex Virus 2 Induces Protective Immunity after Genital Infection

Herpes simplex virus 2 (HSV-2) infects the genital mucosa and establishes a life-long infection in sensory ganglia. After primary infection HSV-2 may reactivate causing recurrent genital ulcerations. HSV-2 infection is prevalent, and globally more than 400 million individuals are infected. As clinical trials have failed to show protection against HSV-2 infection, new vaccine candidates are warranted. The secreted glycoprotein G (sgG-2) of HSV-2 was evaluated as a prophylactic vaccine in mice using two different immunization and adjuvant protocols. The protocol with three intramuscular immunizations combining sgG-2 with cytosine-phosphate-guanine dinucleotide (CpG) motifs and alum induced almost complete protection from genital and systemic disease after intra-vaginal challenge with HSV-2. Robust immunoglobulin G (IgG) antibody titers were detected with no neutralization activity. Purified splenic CD4+ T cells proliferated and produced interferon-γ (IFN-γ) when re-stimulated with the antigen in vitro. sgG-2 + adjuvant intra-muscularly immunized mice showed a significant reduction of infectious HSV-2 and increased IFN-γ levels in vaginal washes. The HSV-2 DNA copy numbers were significantly reduced in dorsal root ganglia, spinal cord, and in serum at day six or day 21 post challenge. We show that a sgG-2 based vaccine is highly effective and can be considered as a novel candidate in the development of a prophylactic vaccine against HSV-2 infection.

Data on the NADPH-oxidase activity induced by WKYMVm and galectin-3 in bone marrow derived and exudated neutrophils isolated from four different mouse strains.

Neutrophils are the key players in inflammatory reactions and the release of superoxide through the NADPH-oxidase upon neutrophil activation contributes to bacterial clearance and surrounding tissue damage. Here we describe data on the mouse neutrophil NADPH-oxidase activation induced by the mouse formyl peptide receptor (Fpr) agonist WKYMVm and galectin-3. Neutrophils isolated from bone marrow, peritoneal exudated, and in vitro TNFα primed bone marrow neutrophils from four different laboratory strains (C57BL/6, DBA/1, BALB/c and NMRI) were used. Both Fpr agonist and galectin-3 activated neutrophils to release superoxide. No differences were observed in the amounts of superoxide released from neutrophils derived from four different strains.

Essential role of galectin3 in reducing the severity of S. aureus-induced arthritis in mice

Background:13C-urea may be a suitable marker to assess the in vivo fate of colon-targeted dosage forms given by mouth. Release in the colon would lead to fermentation of13C-urea into13CO2and excretion of13CO2in breath, release in the small intestine to detection of13C-urea in blood and urine. The differential kinetics of13C-urea can potentially describe both release kinetics and the gastrointestinal segment of release. Materials and methods: Experiments were performed in healthy volunteers.13C urea was administered orally in a coated capsule designed for colon delivery or in an uncoated capsule. Also the use of a second, uncoated capsule filled with15N2-urea was investigated. The recovery of fermented 13C -urea was calculated from breath13C enrichment, the recovery of absorbed 13C-urea from the13C- and15N-enrichments in blood and/or urine. Results: After intake of the colon delivery device, recoveries of13C in breath CO2and in blood as13C-urea showed a high inverse correlation. Total recovery of13C averaged >95%, indicating complete recovery.13CO2exhalation was observed in all subjects. The recovery of13C in urine correlated well with that in blood. The recoveries in urine of13C and15N were identical (mean ratio 1.03) when13C-urea is administered in uncoated capsules and